Genes involved in complex adaptive processes tend to have highly conserved upstream regions in mammalian genomes

BACKGROUND:Recent advances in genome sequencing suggest a remarkable conservation in gene content of mammalian organisms. The similarity in gene repertoire present in different organisms has increased interest in studying regulatory mechanisms of gene expression aimed at elucidating the differences in phenotypes. In particular, a proximal promoter region contains a large number of regulatory elements that control the expression of its downstream gene. Although many studies have focused on identification of these elements, a broader picture on the complexity of transcriptional regulation of different biological processes has not been addressed in mammals. The regulatory complexity may strongly correlate with gene function, as different evolutionary forces must act on the regulatory systems under different biological conditions. We investigate this hypothesis by comparing the conservation of promoters upstream of genes classified in different functional categories.RESULTS:By conducting a rank correlation analysis between functional annotation and upstream sequence alignment scores obtained by human-mouse and human-dog comparison, we found a significantly greater conservation of the upstream sequence of genes involved in development, cell communication, neural functions and signaling processes than those involved in more basic processes shared with unicellular organisms such as metabolism and ribosomal function. This observation persists after controlling for G+C content. Considering conservation as a functional signature, we hypothesize a higher density of cis-regulatory elements upstream of genes participating in complex and adaptive processes.CONCLUSION:We identified a class of functions that are associated with either high or low promoter conservation in mammals. We detected a significant tendency that points to complex and adaptive processes were associated with higher promoter conservation, despite the fact that they have emerged relatively recently during evolution. We described and contrasted several hypotheses that provide a deeper insight into how transcriptional complexity might have been emerged during evolution.

The ecology of the European hedgehog (Erinaceus europaeus) in rural Ireland

This study on the ecology of Irish hedgehogs (Erinaceus europaeus) has provided information on detection techniques, home range, habitat selection, hedgehog prey, nesting, courtship, genetics, road mortality, parasites, ageing and morphology of this species. Data were obtained from a focal study area in rural Cork, in which 24 radio tagged hedgehogs were monitored from June 2008 to June 2010. Further data were obtained through road kill surveys and the collection of hedgehog carcasses from around Ireland. Hedgehogs of both sexes were found to display philopatry. Habitat was not used in proportion to its availability, but certain habitats were selected and a similar pattern of habitat selection was evident in successive years. Hedgehogs preferred arable land in September and October and, unlike studies elsewhere, were observed to forage in the centre of fields. Badgers were regularly seen at the study site and did not appear to negatively affect hedgehogs’ use of the area. Instead the intra- and inter-habitat distribution of hedgehogs was closely correlated with that of their potential prey. Male hedgehogs had a mean annual home range of 56 ha and females 16.5 ha, although monthly home ranges were much more conservative. Male home range peaked during the breeding season (April-July) and a peak in road deaths was observed during these months. The majority of road kill (54%) were individuals of one year old or less, however, individuals were found up to eight and nine years of age. Genetic analysis showed a distinct lack of genetic variation amongst Irish hedgehogs and suggests colonisation by a small number of individuals. The ectoparasites, Archaeopsylla erinacei, Ixodes hexagonus and Ixodes canisuga were recorded in addition to the endoparasites Crenosoma striatum and Capillaria erinacei. In light of the reported decline in many areas of the hedgehogs’ range, it is a species of conservation concern, and this is the first study examining the ecology of the hedgehog in Ireland.

Identification and characterization of innate immune receptor substrates of γ-secretase enzyme complex

The γ-secretase protease complexes and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signalling events, which have a central role in Alzheimer’s disease, cancer progression and immune surveillance. It has previously been reported that the Interleukin-1 receptor, type 1, (IL-1R1) is a substrate for regulated intramembrane proteolysis, mediated by presenilin (PS)-dependent γ-secretase activity. The aims of this project were twofold. Firstly, to determine the conservation of regulated intramembrane proteolysis as a physiological occurrence amongst other cytokine receptors. In this regard, similar to IL-1R1, we identified the Tumour necrosis factor receptor type 1 (TNFR1) and the Toll like receptor 4 (TLR4) as novel γ-secretase substrates. Secondly, given that the diversity of signalling events mediated by the IL-1R1, TLR4 and TNFR1 are spatially segregated, we investigated the spatial distribution, subcellular trafficking and subcellular occurrence of regulated intramembrane proteolysis of IL-1R1, TLR4 and TNFR1. Using dynasore an inhibitor of clathrin-dependent receptor endocytosis, both ectodomain shedding and γ-secretase-mediated cleavage of IL-1R1 were observed post-internalization. In contrast, TNFR-1 underwent ectodomain shedding at the cell surface followed by endosomal γ-secretase-mediated cleavage. Furthermore, immortalised fibroblasts from PS1-deficient mice showed impaired γ-secretasemediated cleavage of IL-1R1 and TNFR1, indicating that both are cleaved by PS1-and not PS2-containing γ-secretase complexes. Subcellular fractionation and immunofluorescence studies revealed that the γ-secretase generated IL-1R1 ICD translocates to the nucleus on IL-1β stimulation. These observations further demonstrate the novel PS-dependent means of modulating IL-1β, LPS and TNFα- mediated immune responses by regulating IL-1R1/TLR4/TNFR1 protein levels within the cells.

Comparing impacts of new cropping systems on biodiversity in traditional rural China

This study selected six geographically-similar villages with traditional and alternative cultivation methods (two groups of three, one traditional and two alternatives) in two counties of Henan Province, China—a representative area of the Huang-huai-hai Plain representing traditional rural China. Soil heavy metal concentrations, floral and faunal biodiversity, and socio-economic data were recorded. Heavy metal concentrations of surface soils from three sites in each village were analysed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS, chromium, nickel, copper, cadmium, and lead) and Atomic Absorption Spectrophotometer (AAS, zinc). The floral biodiversity of four land-use types was recorded following the Braun-Blanquet coverage-abundance method using 0.5×0.5m quadrats. The faunal biodiversity of two representative farmland plots was recorded using 0.3×0.3m quadrats at four 0.1m layers. The socio-economic data were recorded through face-to-face interviews of one hundred randomly selected households at each village. Results demonstrate different cultivation methods lead to different impact on above variables. Traditional cultivation led to lower heavy metal concentrations; both alternative managements were associated with massive agrochemical input causing heavy metal pollution in farmlands. Floral distribution was significantly affected by village factors. Diverse cultivation supported high floral biodiversity through multi-scale heterogeneous landscapes containing niches and habitats. Faunal distribution was also significantly affected by village factor nested within soil depth. Different faunal groups responded differently, with Acari being taxonomically diverse and Collembola high in densities. Increase in manual labour and crop number in villages using alternative cultivation may positively affect biodiversity. The results point to the conservation potential of diverse cultivation methods in traditional rural China and other regions under social and political reforms, where traditional agriculture is changing to unified, large-scale mechanized agriculture. This study serves as a baseline for conservation in small-holding agricultural areas of China, and points to the necessity of further studies at larger and longer scales.

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Lack of cross-scale linkages reduces robustness of community-based fisheries management.

Community-based management and the establishment of marine reserves have been advocated worldwide as means to overcome overexploitation of fisheries. Yet, researchers and managers are divided regarding the effectiveness of these measures. The "tragedy of the commons" model is often accepted as a universal paradigm, which assumes that unless managed by the State or privatized, common-pool resources are inevitably overexploited due to conflicts between the self-interest of individuals and the goals of a group as a whole. Under this paradigm, the emergence and maintenance of effective community-based efforts that include cooperative risky decisions as the establishment of marine reserves could not occur. In this paper, we question these assumptions and show that outcomes of commons dilemmas can be complex and scale-dependent. We studied the evolution and effectiveness of a community-based management effort to establish, monitor, and enforce a marine reserve network in the Gulf of California, Mexico. Our findings build on social and ecological research before (1997-2001), during (2002) and after (2003-2004) the establishment of marine reserves, which included participant observation in >100 fishing trips and meetings, interviews, as well as fishery dependent and independent monitoring. We found that locally crafted and enforced harvesting rules led to a rapid increase in resource abundance. Nevertheless, news about this increase spread quickly at a regional scale, resulting in poaching from outsiders and a subsequent rapid cascading effect on fishing resources and locally-designed rule compliance. We show that cooperation for management of common-pool fisheries, in which marine reserves form a core component of the system, can emerge, evolve rapidly, and be effective at a local scale even in recently organized fisheries. Stakeholder participation in monitoring, where there is a rapid feedback of the systems response, can play a key role in reinforcing cooperation. However, without cross-scale linkages with higher levels of governance, increase of local fishery stocks may attract outsiders who, if not restricted, will overharvest and threaten local governance. Fishers and fishing communities require incentives to maintain their management efforts. Rewarding local effective management with formal cross-scale governance recognition and support can generate these incentives.

The Emergence of Access Controls in Small-Scale Fishing Commons: A Comparative Analysis of Individual Licenses and Common Property-Rights in Two Mexican Communities

Addressing global fisheries overexploitation requires better understanding of how small-scale fishing communities in developing countries limit access to fishing grounds. We analyze the performance of a system based on individual licenses and a common property-rights regime in their ability to generate incentives for self-governance and conservation of fishery resources. Using a qualitative before-after-control-impact approach, we compare two neighbouring fishing communities in the Gulf of California, Mexico. Both were initially governed by the same permit system, are situated in the same ecosystem, use similar harvesting technology, and have overharvested similar species. One community changed to a common property-right regime, enabling the emergence of access controls and avoiding overexploitation of benthic resources, while the other community, still relies on the permit system. We discuss the roles played by power, institutions, socio-historic, and biophysical factors to develop access controls. © 2012 The Author(s).

English Channel towed sledge seabed images. Phase 2: Analysis of selected tow images

During the 1970s and 1980s, the late Dr Norman Holme undertook extensive towed sledge surveys in the English Channel and some in the Irish Sea. Only a minority of the resulting images were analysed and reported before his death in 1989 but logbooks, video and film material has been archived in the National Marine Biological Library (NMBL) in Plymouth. A study was therefore commissioned by the Joint Nature Conservation Committee and as a part of the Mapping European Seabed Habitats (MESH) project to identify the value of the material archived and the procedure and cost to undertake further work (Phase 1 of the study reported here: Oakley & Hiscock, 2005). Some image analysis was undertaken as a part of Phase 1. Phase 2 (this report) was to further analyse selected images. Having determined in Phase 1 that only the 35 mm photographic transparencies provided sufficient clarity to identify species and biotopes, the tows selected for analysis were ones where 35mm images had been taken. The tows selected for analysis of images were mainly in the vicinity of Plymouth and especially along the area between Rame Head and the region of the Eddystone. The 35 mm films were viewed under a binocular microscope and the taxa that could be recognised recorded in note form. Twenty-five images were selected for inclusion in the report. Almost all of the images were of level sediment seabed. Where rocks were included, it was usually unplanned and the sled was hauled before being caught or damaged. The main biotopes or biotope complexes identified were: SS.SMU.CSaMu. Circalittoral sandy mud. Extensively present between the shore and the Eddystone Reef complex and at depths of about 48 to 52 m. At one site offshore of Plymouth Sound, the turret shell Turritella communis was abundant. In some areas, this biotope had dense anemones, Mesacmaea mitchelli and (more rarely) Cerianthus lloydii. Queen scallops, Aequipecten opercularis and king scallops, Pecten maximus, were sometimes present in small numbers. Hard substratum species such as hydroids, dead mens fingers Alcyonium digitatum and the cup coral Caryophyllia smithii occurred in a few places, probably attached to shells or stones beneath the surface. South of the spoil ground off Hilsea Point at 57m depth, the sediment was muddier but is still assigned to this biotope complex. It is notable that three small sea pens, most likely Virgularia mirabilis, were seen here. SS.SMx.CMx. Circalittoral mixed sediment. Further offshore but at about the same depth as SS.SMU.CSaMu occurred, coarse gravel with some silt was present. The sediment was characterised must conspicuously by small queen scallops, Aequipecten opercularis. Peculiarly, there were ‘bundles’ of the branching bryozoan Cellaria sp. – a species normally found attached to rock. It could not be seen whether these bundles of Cellaria had been brought-together by terebellid worms but it is notable that Cellaria is recorded in historical surveys. As with many other sediments, there were occasional brittle stars, Ophiocomina nigra and Ophiura ophiura. Where sediments were muddy, the burrowing anemone Mesacmaea mitchelli was common. Where pebbles or cobbles occurred, there were attached species such as Alcyonium digitatum, Caryophyllia smithii and the fleshy bryozoan Alcyonidium diaphanum. Undescribed biotope. Although most likely a part of SS.SMx.CMx, the biotope visually dominated by a terebellid worm believed to be Thelepus cincinnatua, is worth special attention as it may be an undescribed biotope. The biotope occurred about 22 nautical miles south of the latitude of the Eddystone and in depths in excess of 70 m. SS.SCS.CCS.Blan. Branchiostoma lanceolatum in circalittoral coarse sand with shell gravel at about 48m depth and less. This habitat was the ‘classic’ ‘Eddystone Shell Gravel’ which is sampled for Branchiostoma lanceolatum. However, no Branchiostoma lanceolatum could be seen. The gravel was almost entirely bare of epibiota. There were occasional rock outcrops or cobbles which had epibiota including encrusting calcareous algae, the sea fan Eunicella verrucosa, cup corals, Caryophyllia smithii, hydroids and a sea urchin Echinus esculentus. The variety of species visible on the surface is small and therefore identification to biotope not usually possible. Historical records from sampling surveys that used grabs and dredges at the end of the 19th century and early 20th century suggest similar species present then. Illustrations of some of the infaunal communities from work in the 1920’s is included in this report to provide a context to the epifaunal photographs.

Predicted levels of future ocean acidification and temperature rise could alter community structure and biodiversity in marine benthic communities

A mesocosm experiment was conducted to quantify the effects of reduced pH and elevated temperature on an intact marine invertebrate community. Standardised faunal communities, collected from the extreme low intertidal zone using artificial substrate units, were exposed to one of eight nominal treatments (four pH levels: 8.0, 7.7, 7.3 and 6.7, crossed with two temperature levels: 12 and 16°C). After 60 days exposure communities showed significant changes in structure and lower diversity in response to reduced pH. The response to temperature was more complex. At higher pH levels (8.0 and 7.7) elevated temperature treatments contained higher species abundances and diversity than the lower temperature treatments. In contrast, at lower pH levels (7.3 and 6.7), elevated temperature treatments had lower species abundances and diversity than lower temperature treatments. The species losses responsible for these changes in community structure and diversity were not randomly distributed across the different phyla examined. Molluscs showed the greatest reduction in abundance and diversity in response to low pH and elevated temperature, whilst annelid abundance and diversity was mostly unaffected by low pH and was higher at the elevated temperature. The arthropod response was between these two extremes with moderately reduced abundance and diversity at low pH and elevated temperature. Nematode abundance increased in response to low pH and elevated temperature, probably due to the reduction of ecological constraints, such as predation and competition, caused by a decrease in macrofaunal abundance. This community-based mesocosm study supports previous suggestions, based on observations of direct physiological impacts, that ocean acidification induced changes in marine biodiversity will be driven by differential vulnerability within and between different taxonomical groups. This study also illustrates the importance of considering indirect effects that occur within multispecies assemblages when attempting to predict the consequences of ocean acidification and global warming on marine communities.

Diversity of functional groups in the vegetation sandy seashore of the Grande, Anclitas and Caguamas Cays (Jardines de la Reina Archipelago, Cuba)

Sandy shores are known to be extreme ecosystems where the vegetation has evolved many morphological and physiological adaptations for its survival. With the aim of identify possible relationships between the vegetation´s functional diversity with abiotic factors and its corresponding quantification, we collected data on the abundance and richness of the sandy coast vegetation complex in Grande, Anclitas and Caguamas keys. Its flora is largely characterized by the dominance of hemicryptophytes and chamaephytes plants with nanophyllous leaves and displaying dispersal syndromes such as zoochory and anemochory. However, the functional groups´ richness, in the present study, varies from one key to another. Functional diversity is similar between the wet and dry seasons, and its spatial variation is influenced by the interplay of the set of abiotic factors herein studied.

Pattern and scale of variability among Northwest Atlantic Shelf plankton communities

Continuous plankton recorders (CPRs) have been used in the Northwest Atlantic for almost 50 years. While data collected by these surveys have provided valuable information on long-term variability in plankton populations, all previous analyses have been limited to only a portion of the geographic range of the available data. Here we present an analysis of the CPR data from the Mid Atlantic Bight to the Labrador Sea. Across this wide geographic range, we found many common associations among the taxa. In particular, the changes in most regions were strongly size structured, with small and medium copepods varying together and often positively related to indicators of phytoplankton abundance. The time series from nearby regions were strongly correlated; however, after 1990, the spatial pattern became more complex. During this period, several of the copepod taxa, noticeably Calanus finmarchicus and Centropages typicus, experienced a series of anomalies that appeared to propagate from northeast to southwest. Although the direction of propagation was consistent with the shelf circulation, the anomalies propagated at a rate much slower than typical current speeds. The timing of the copepod anomalies and their phase speed were similar in character to observed changes in salinity and the position of the Shelf Slope Front. The correspondence between the changes in the plankton community and changes in the physical environmental suggests that physical conditions are a strong driver of interannual variability in Northwest Atlantic Shelf ecosystems.

Ecohydrodynamics of cold-water coral reefs: A case study of the Mingulay Reef complex (western Scotland)

Ecohydrodynamics investigates the hydrodynamic constraints on ecosystems across different temporal and spatial scales. Ecohydrodynamics play a pivotal role in the structure and functioning of marine ecosystems, however the lack of integrated complex flow models for deep-water ecosystems beyond the coastal zone prevents further synthesis in these settings. We present a hydrodynamic model for one of Earth's most biologically diverse deep-water ecosystems, cold-water coral reefs. The Mingulay Reef Complex (western Scotland) is an inshore seascape of cold-water coral reefs formed by the scleractinian coral Lophelia pertusa. We applied single-image edge detection and composite front maps using satellite remote sensing, to detect oceanographic fronts and peaks of chlorophyll a values that likely affect food supply to corals and other suspension-feeding fauna. We also present a high resolution 3D ocean model to incorporate salient aspects of the regional and local oceanography. Model validation using in situ current speed, direction and sea elevation data confirmed the model's realistic representation of spatial and temporal aspects of circulation at the reef complex including a tidally driven current regime, eddies, and downwelling phenomena. This novel combination of 3D hydrodynamic modelling and remote sensing in deep-water ecosystems improves our understanding of the temporal and spatial scales of ecological processes occurring in marine systems. The modelled information has been integrated into a 3D GIS, providing a user interface for visualization and interrogation of results that allows wider ecological application of the model and that can provide valuable input for marine biodiversity and conservation applications.

On the Front Line: frontal zones as priority at-sea conservation areas for mobile marine vertebrates

1.Identifying priority areas for marine vertebrate conservation is complex because species of conservation concern are highly mobile, inhabit dynamic habitats and are difficult to monitor. 2.Many marine vertebrates are known to associate with oceanographic fronts – physical interfaces at the transition between water masses – for foraging and migration, making them important candidate sites for conservation. Here, we review associations between marine vertebrates and fronts and how they vary with scale, regional oceanography and foraging ecology. 3.Accessibility, spatiotemporal predictability and relative productivity of front-associated foraging habitats are key aspects of their ecological importance. Predictable mesoscale (10s–100s km) regions of persistent frontal activity (‘frontal zones’) are particularly significant. 4.Frontal zones are hotspots of overlap between critical habitat and spatially explicit anthropogenic threats, such as the concentration of fisheries activity. As such, they represent tractable conservation units, in which to target measures for threat mitigation. 5.Front mapping via Earth observation (EO) remote sensing facilitates identification and monitoring of these hotspots of vulnerability. Seasonal or climatological products can locate biophysical hotspots, while near-real-time front mapping augments the suite of tools supporting spatially dynamic ocean management. 6.Synthesis and applications. Frontal zones are ecologically important for mobile marine vertebrates. We surmise that relative accessibility, predictability and productivity are key biophysical characteristics of ecologically significant frontal zones in contrasting oceanographic regions. Persistent frontal zones are potential priority conservation areas for multiple marine vertebrate taxa and are easily identifiable through front mapping via EO remote sensing. These insights are useful for marine spatial planning and marine biodiversity conservation, both within Exclusive Economic Zones and in the open oceans.

Effect of habitat fragmentation on the macroinvertebrate infaunal communities associated with the seagrass Zostera marina

1. The effect of habitat fragmentation was investigated in two adjacent, yet separate, intertidal Zostera marina beds in the Salcombe Estuary, Devon, UK. The seagrass bed on the west bank comprised a continuous meadow of ca. 2.3 ha, whilst the bed on the east bank of the estuary was fragmented into patches of 6–9 m2.2. Three 10 cm diameter core samples for infaunal macroinvertebrates were taken from three stations within each bed. No significant difference was found in univariate community parameters between beds, or in measured seagrass parameters. However, multivariate analysis revealed a significant difference in community composition, due mainly to small changes in species abundance rather than differences in the species present.3. The species contributing most to the dissimilarity between the two communities were polychaetes generally associated with unvegetated habitats (e.g. Magelona mirabilis) and found to be more common in the fragmented bed.4. A significant difference in median grain size and sorting coefficient was recorded between the two beds, and median grain size was found to be the variable best explaining multivariate community patterns.5. The results of the study provide evidence for the effects of habitat fragmentation on the communities associated with seagrass beds, habitats which are of high conservation importance. As the infaunal community is perhaps intuitively the component least likely to be affected by fragmentation at the scale observed, the significant difference in community composition recorded has consequences for more sensitive and high-profile parts of the biota (e.g. fish), and thus for the conservation of seagrass habitats and their associated communities.

Assessing the conservation status of marine habitats: thoughts from a sandflat on the Isles of Scilly.

Statutory monitoring of the fauna of the ‘mudflats and sandflats not covered by seawater at low tide’ biotope complex on St Martin’s Flats, a part of the Isles of Scilly Complex Special Area of Conservation, was undertaken in 2000, 2004 and 2009. The targets set by Natural England for “characteristic biotopes” were that “composite species, abundance and diversity should not deviate significantly from an established baseline, subject to natural change”. The three specified biotopes could not be distinguished, and instead three assemblages were subjectively defined based on sediment surface features. There were statistically significant natural changes in diversity and species composition between years, especially in the association initially characterized by the razor-clam Ensis, and possible reasons for this are discussed. It is suggested that setting fixed local limits on natural variability is almost always impractical. Two possible approaches to distinguishing between natural and anthropogenic changes are suggested; a change in ecological condition as indicated by AMBI scores, and a significant change in average taxonomic distinctness (Δ+) compared with expectation. The determination of species biomasses as well as abundances might also open more possibilities for assessment. The practice of setting objectives for a marine SAC feature that include the range and number of biotopes cannot be supported, in view the difficulty in ascribing assemblages to recognised biotopes. A more realistic definition of species assemblages might best be gained from examination of the species that consistently make a substantial contribution to the Bray Curtis similarity among samples collected from specific sites.